Microbial Genomics

Microbial Genomics : 

Microbial Genomics M Adnan Ashraf

Introduction: 

Introduction Genomics is the study of the molecular organization of genomes, their information content, and the gene products they encode. It helps us understand microbial physiology of organisms. It helps us understand evolution of all organisms. Categories: Structural Genomics Functional Genomics Comparative Genomics

Structural Genomics: 

Structural Genomics Study to determine and analyze the DNA sequence of the genome.

Determining DNA Sequences: 

Determining DNA Sequences Sanger’s Technique: Sanger’s technique is also known as Chain-termination DNA sequencing method. Single strands of template DNA are mixed with primer, DNA polymerase, the four deoxynucleoside triphosphates ( dNTPs ), and one of the dideoxynucleoside triphosphates ( ddNTPs ). Four such reactions are prepared, one for each ddNTP .

Determining DNA Sequences: 

Determining DNA Sequences

Determining DNA Sequences: 

Determining DNA Sequences ddNTPs differ from dNTPs in that the 3’ carbon lacks hydroxyl group.

Determining DNA Sequences: 

Determining DNA Sequences

Determining DNA Sequences: 

Determining DNA Sequences

Determining DNA Sequences: 

Determining DNA Sequences DNA synthesis will stop when a ddNTP is added in the growing chain. Reaction stops because the 3’-OH group will not be available to attack the 5’-PO4 of the next dNTP. DNA fragments of varying length are generated in each reaction mixture when reaction stops.

Determining DNA Sequences: 

Determining DNA Sequences

Determining DNA Sequences: 

Determining DNA Sequences Manual Sequencing: To manually sequence, radioactive dNTPs are used. Each reaction is electrophoresed in a separate lane on a polyacrylamide gel. Shorter fragments migrate faster. ddNTP at the end of shortest fragment is assigned 5' end of the DNA sequence. While the largest fragment if assigned the 3' end. So, DNA sequence is read from smallest to the largest fragment.

Manual Sequencing: 

Manual Sequencing

Determining DNA Sequences: 

Determining DNA Sequences Automated Sequencing: All the four reaction mixtures are loaded into a single lane of a gel. Each ddNTP is labeled with different fluorescent dye. Fragments produced are electrophoresed on a polyacrylamide gel.

Automated Sequencing: 

Automated Sequencing

Automated Sequencing: 

Automated Sequencing

Automated Sequencing: 

Automated Sequencing Laser beam determines the order of their exit. A chromatogram is generated, whose amplitudes tell the sequence of DNA. One million bases per day can be sequenced by these sequencers. Many sequencers running at the same time are needed for whole genome.

Automated Sequencing: 

Automated Sequencing

Automated Sequencing: 

Automated Sequencing

Whole-Genome Shotgun Sequence: 

Whole-Genome Shotgun Sequence It comprises four stages. Library Construction: DNA molecules are randomly broken down into small fragments using ultrasonic waves. Then these are purified.

Library Construction: 

Library Construction

Library Construction: 

Library Construction

Library Construction: 

Library Construction These fragments are then attached to plasmid to get a single insert. Special E coli strains are transformed with the plasmids to produce a library of plasmid clones.

Library Construction: 

Library Construction

Whole-Genome Shotgun Sequence: 

Whole-Genome Shotgun Sequence Random Sequencing: Thousands of DNA fragments are sequenced with automated sequencers. Almost all stretches of genome are sequenced several times.

Whole-Genome Shotgun Sequence: 

Whole-Genome Shotgun Sequence Fragment Alignment and Gap closure: Sequenced DNA fragment data is assembled into longer stretches using special computer programs. Two fragments are joined together if their ends overlap and match, resulting in a set of larger nucleotide sequences called contigs . Contigs are aligned in proper order to complete genome sequence.

Alignment of Contigs: 

Alignment of Contigs

Whole-Genome Shotgun Sequence: 

Whole-Genome Shotgun Sequence If there is a gap between two contigs , Fragments with ends in two adjacent contigs are located. Gaps are filled by analyzing these fragments. Gap sequence can also be obtained from libraries containing large DNA fragments. Editing: Sequnce is proofread.

Gap Closure: 

Gap Closure

Functional Genomics: 

Functional Genomics Study to examine the transcripts and array of proteins produced by the genome.

Microarray Analysis: 

Microarray Analysis

Microarray Analysis: 

Microarray Analysis Microarray analysis allows us to look at the vast collection of gene expression at once. DNA microarrays are solid supports of glass or silicon upon which DNA is attached. DNA is attached at specific spots. Each spot of DNA represents a single gene called a probe. Microarray contains hundreds of thousands of probes. Analysis of gene expression is based on hybridization between the probe DNA and the targets. Target is mRNA or cDNA to be tested.

Microarray Analysis: 

Microarray Analysis

Microarray Analysis: 

Microarray Analysis To determine the change in expression of genes in response to environmental changes. Total cellular mRNA exposed to environmetal changes is labeled red . (experimental) Total cellular mRNA grown in neutral conditions is prepared and tagged with green fluorochrome. (reference) Reference and experimental samples are mixed to the same microarray in equal quantities.

Microarray Analysis: 

Microarray Analysis

Microarray Analysis: 

Microarray Analysis

Microarray Analysis: 

Microarray Analysis

Microarray Analysis: 

Microarray Analysis Unattached mRNA is washed off, chip is scanned and image is computer analyzed. Red spot indicates gene was induced. Green spot indicates gene was repressed. Yellow spot indicates, there was no change in level of gene expression of gene. Black spot indicates neither the sample nor the controlled nuclear material is hybridized.

Microarray Analysis: 

Microarray Analysis

Microarray Analysis: 

Microarray Analysis Limitations: Results only represent mRNA present at the time of preparation. Some of posttranslational mRNA will be present but protein may not be active.

Comparative Genomics: 

Comparative Genomics Study to compare genomes to look for differences and similarities. Genomes are compared to study evolution and speciation. Genomes are compared to determine the evolution of pathogenicity. Comparative genomics is applied in the development of vaccines. Genomes of all pathogenic strains are compared for a suitable antigen to make vaccines.

Proteomics: 

Proteomics The entire collection of proteins that an organism produces is called its proteome. Study of array of proteins of an organism is called proteomics. Proteomics provide information about genome function that mRNA studies cannot. mRNA level tells what might occur in the cell, whereas proteomics tells what is actually happening in the cell.

Two-dimensional Gel Electrophoresis : 

Two-dimensional Gel Electrophoresis

Two-dimensional Gel Electrophoresis : 

Two-dimensional Gel Electrophoresis Mixture of proteins is separated using two electrophoretic procedures (dimensions). First dimension is polyacrylamide gel set in a tube. In first dimension proteins move electrophoretically through a pH gradient until protein’s net charge is zero.

First Dimension Electrophoresis: 

First Dimension Electrophoresis

2nd Dimension Electrophoresis: 

2 nd Dimension Electrophoresis Second dimension is SDS-PAGE. Gel from first dimension is soaked in SDS buffer.

Soaking in SDS: 

Soaking in SDS

2nd Dimension Electrophoresis: 

2 nd Dimension Electrophoresis Then gel of first dimension is placed at the edge of second dimension gel. Voltage is applied and polypeptides are separated according to their molecular weight. Smallest polypeptide travels fastest and farthest. Each protein is visualised as a separate spot.

2nd Dimension Electrophoresis: 

2 nd Dimension Electrophoresis

2nd Dimension Electrophoresis: 

2 nd Dimension Electrophoresis 2-D gel is used to compare microbes grown under different conditions.

2-D Gel Electrophoresis: 

2-D Gel Electrophoresis

References : 

References Joanne M Willey, Linda M Sherwood, ‘ Prescott's Principles of Microbiology’ , Boston [u.a.] McGraw-Hill 2008